I. Field of Invention
The present invention relates to a method of detecting gene expression in cells and, if desired, of preventing such expression. More particularly, the invention relates to methods of detecting mRNA in the presence of complementary genomic DNA, and of preventing the expression of such complementary DNA. The invention also relates to specific testing or identification procedures using such methods.
II. Description of the Prior Art
Specific gene expression or the lack thereof can be indicative of a genetic defect, a disease marker or a viral infection. Therefore, it is routinely desirable to investigate the expression of specific genes. mRNA is produced in cells during gene translation to form a corresponding protein, so the detection of a particular mRNA in a cell is evidence of activation of the corresponding gene. In other words, if mRNA is present in a cell, it may be assumed that gene expression is taking place.
It is possible to detect mRNA by means of the polymerase chain reaction (PCR) that is used to amplify the miniscule amounts of mRNA present to provide quantities suitable for detection and/or identification. This is possible because PCR is capable of achieving amplification rates in excess of a millionfold.
The protocol for the polymerase chain reaction has been known for several years and has achieved widespread application in the fields of medical diagnostics and forensics. U.S. Pat. Nos. 4,683,202 and 4,683,195, for example, describe the original PCR process for the amplification and detection of nucleic acid sequences. The basic principle of PCR relies on the repetition of the following steps:
1) Denaturation--the template strands of the originating DNA sample are subjected to elevated temperatures and are subsequently denatured to form single stranded DNA templates. PA1 2) Renaturation--oligonucleotide primers complementary to regions flanking a gene or other DNA sequence of interest are hybridized to the single stranded DNA templates at the 3'-ends of the template sequence at an optimally lower temperature. PA1 3) Synthesis--thermostable DNA polymerase present in the sample mixture functions to extend the oligonucleotide primers from the 3'-ends with nucleotide triphosphates complimentary to the template nucleotide sequence. PA1 4) Repetition--the above steps are repeated many times. Each time the denaturation step is completed, DNA strands newly formed in the preceding step are released as single stranded DNA templates for the subsequent steps, thus increasing the copies of the sequence of interest exponentially as the repetitions progress. PA1 1) it limits the use of this technique for routine medical application which require more rapid results; and PA1 2) it adds an element of uncertainty as the procedure lacks an indicator of complete removal of the genomic DNA. PA1 1) Fixation of cells or tissues--isolate target cell/tissue for amplification/identification. PA1 2) Pepsin digestion--disruption of cytoplasmic membrane, making holes for the reagents to enter the cells, while retaining the integrity of the cell wall. PA1 3) Reverse transcription--construct an mRNA derived cDNA template. PA1 4) Polymerase reaction--wherein the PCR mixture includes modified oligonucleotides as intron-blockers and PCR primers, the primers preferably incorporating a fluorescent label or other identification means. PA1 5) Detection by visualizing a fluorescent signal or by other means. Positive detection indicates the presence of mRNA and thus confirms gene expression. Depending on the particular test, this may be indicative of a genetic defect, a specific protein, viral infection, disease specific marker, etc.
With continued use, the basic PCR technique has been modified or extended in various ways. Advancements in the field of PCR technology are described, for example, in U.S. Pat. Nos. 5,436,149, 5,405,774, 5,340,728 and 5,338,671.One of the ways in which PCR has been extended is to embrace the use of mRNA as a template for nucleotide sequence amplification. This is discussed, for example, in U.S. Pat. Nos. 5,407,800, 5,322,770 and 5,527,669, wherein cDNA is first synthesized from an mRNA template by reverse transcription and subsequently amplified by PCR. Reliant on the activity of an enzyme, reverse transcriptase (RT), deoxyribonucleotides complementary to an mRNA template are directed into a growing cDNA strand.
In-situ PCR techniques involving reverse transcriptase (RT-PCR) have been developed to detect levels of particular mRNAs in cells. However, limitations have been encountered with this approach when the primer pair introduced to initiate amplification of the cDNA template also amplifies the corresponding region of the genome, thus generating false positive results. The detection of a gene in the genome does not, of course, mean that the gene is being expressed, but merely that it is present.
In attempt to overcome this imprecision, techniques have been adopted to eliminate the genomic DNA by digestion in the presence of a DNase enzyme prior to initiation of reverse transcription. This digestion step, as presently used, takes at least seven hours for completion and creates additional problems, such as the following:
There is therefore a need for an improved procedure for the detection of gene expression involving in-situ PCR to overcome the false positive problem while avoiding disadvantages of known procedures, such as those mentioned above.